This invention relates generally to constructs used in tissue-engineering, and more particularly, to cell delivery constructs comprising a controllable degradable gel phase, meshed within a polymer substrate.
The field of tissue engineering has brought recent advances to the development of substitutes for donor tissue and improved methods of tissue repair. For example, by incorporating principles from the materials and biological sciences, constructs can now be designed to culture functional tissue in vitro for use in subsequent surgical implantation. See Langer and Vacanti, Science 260:920, 1993. These constructs often require a biocompatible support substrate to allow cells to be seeded and grown. By facilitating new tissue growth, these constructs offer a potential to ameliorate the limited supply of donor tissue and also, improve the resources available for repairing damaged and non-functional tissue.
The use of biocompatible, biodegradable, polymer substrates in tissue engineering constructs for seeding cells and growing new tissue, has been demonstrated by Langer and others using a variety of cell types. See Cima and Langer, Chem. Eng. Prog. 46, 1993; Takeda et al. Transplantation Proc. 27(1):635. The ability to incorporate different cell types greatly expands the versatility and applicability of such a device for use in various medical procedures. For example, scaffolds can be designed for applications ranging from the development of new muscle tissue, to the production of new arteries. See Niklason and Langer, Transplant Immunology 5:303, 1997. One example of a tissue engineering device is described in co-pending U.S. patent application Ser. No. 09/109,427 incorporated herein by reference. One embodiment of that invention is a device comprising a tubular substrate onto which muscle cells can be seeded and grown. The substrate is porous and comprised of a biodegradable polymer such as polyglycolic acid (PGA), polylactic acid (PLA), or poly lactic-co-glycolic acid (PLGA).
Although PGA scaffolds have a proven clinical history and offer certain advantages for use in tissue engineering, one major disadvantage is their lack of structural integrity immediately after the cell seeding process. In particular, the scaffold is xe2x80x9cwetxe2x80x9d with media and cells and remains very fragile for several weeks following seeding. However, implantation of cell scaffold units immediately after seeding facilitates quick integration of newly forming tissue with the existing tissue, thus improving the available methods of tissue engineering and repair.
Once the device is implanted, the seeded cells quickly begin to proliferate and release extracellular matrix molecules into the surrounding tissue. Therefore, it is also advantageous to have a device which can deliver a high concentration or density of cells. An example of a surgical application benefiting from immediate implantation of a freshly seeded scaffold is in the repair and replacement of cartilage tissue and specifically, the meniscal tissue of the knee.
Immediate implantation, however, requires that the device have the mechanical strength to withstand the implantation and suturing procedures directly after seeding the cells. Thus, the lack of integrity of conventional freshly seeded scaffolds, renders them inoperative for use in many medical and surgical applications.
One approach to develop a biocompatible device for delivering cells directly after seeding has been to entrap the cells within a gel. Although this method provides the ability to achieve high cell densities, it has two major limitations. First, most gels are similar to PGA meshes in that they lack mechanical strength. The gel entrapment device is not rigid and is therefore unable to withstand the implantation and suturing procedures. Attempts have been made to increase the strength of gel entrapment constructs (e.g. using gel solidification techniques such as photopolymerization), but these techniques are often harsh and result in damage or death to the cells. Second, gel entrapment constructs can hinder the immediate growth and proliferation of cells.
Without a means to control the degradation of the gel, the growth and matrix production of the cells is limited. The need therefore remains for a construct that can deliver a high concentration of cells, while retaining the structural integrity to withstand surgical procedures. The controllable degradable mesh-gel constructs of the present invention provide such a construct.
According to one aspect, the invention is a cell delivery construct comprising a gel phase meshed within a polymer substrate. The gel phase comprises a degradable, natural or synthetic polymer, and includes a suspension of living cells. The gel phase for example, could comprise polymerized fibrin, polymerized heparin, glycosaminoglycans, sugars, polysaccharides, self assembling peptides, or proteins. In a preferred embodiment, the gel phase comprises polymerized fibrin.
The polymer substrate comprises a biocompatible, degradable polymer, and may be synthetic or natural. A synthetic polymer may be selected from the group consisting of polyglycolic acid (PGA), polylactic acid (PLA), or poly lactic-co-glycolic acid (PLGA). In one embodiment, the polymer substrate comprises PGA. A natural polymer substrate may comprise collagen, alginic acid, cellulose, silk, starch, or pullalan. In one embodiment, the natural polymer comprises collagen.
The gel phase includes a suspension of living cells, whose selection depends on the desired application of the end construct. In one embodiment, the cells comprise cartilage tissue cells and have a final density of 5xc3x97106 cells/construct, the construct having a diameter of 0.5 cm, and a thickness of 0.2 cm.
In accordance with the invention, the cell delivery construct allows for the controlled degradation of the gel phase. The gel phase may be degraded with enzymes or by adjusting the physical properties of the gel phase, such as pH, temperature, or ionic equilibrium. Urokinase and heparinase are suitable enzymes for degrading a gel phase comprising polymerized fibrin and heparin respectively. In preferred embodiments, the enzyme is either solubilized in the gel phase or encapsulated within biocompatible, biodegradable, polymer microspheres.
In another embodiment, cross-linkers or inhibitors are solubilized in the gel phase or encapsulated within biocompatible, biodegradable, polymer microspheres. By hindering or slowing degradation, the cross-linkers or inhibitors provide further control over the degradation process.
According to another aspect, the invention is a method of producing a cell delivery construct. The steps of the method comprise providing a degradable gel phase including a suspension of living cells, providing a biocompatible, degradable, polymer substrate, and meshing the gel phase within the polymer substrate.